Coordinator: José Rino (IMM)
Date: September 7-10, 2011
Location: Instituto de Medicina Molecular, Lisbon

The practical component of the Workshop will consist of two modules designed to complement each other:

• Quantitative live cell image acquisition
• Digital image processing and analysis

The first module aims to be a practical guide for quantitative fluorescent imaging of living cells, covering the most relevant aspects from choice of equipment to optimization of acquisition parameters in live cell experiments in order to achieve a compromise between image quality and spatio-temporal resolution while minimizing photobleaching and phototoxicity. A special emphasis will be placed on how to design experiments that yield digital images amenable to automated image analysis.

The second module will introduce the basic concepts of digital image processing in quantitative light microscopy. Students will work with digital images acquired in the first module. The following topics will be covered: gray level and binary operations, image measurement, point spread function/deconvolution, detection/segmentation, localization and particle tracking/trajectory construction.

Program Outline:

The first module will consist of two days of hands-on laboratory exercises performed with small groups of 3 students. A total of 5 microscopes will be used, equipped with cage or chamber incubators for temperature and CO2 control, 1 spinning disk confocal system with motorized stages and piezo, 1 line-scanning confocal unit, 2 point-scanning confocal units for Fluorescence Recovery After Photobleaching (FRAP) and Förster Resonance Energy Transfer (FRET) and 1 cooled CCD fluorescence widefield motorized system.

Laboratories :

• 1a – PSF Widefield microscope - small beads in water, low and high NA objectives (2h – 3 students)
• 1b – PSF Confocal Microscope – small beads in water, low and high NA objectives (2h – 3 students)

• 2a – 2D time-lapse imaging – live cells with malaria parasite – Widefield microscope (2h – 2 students)
• 2b – 2D time-lapse imaging – live cells with malaria parasite – Spinning Disk 1 (2h – 2 students)

• 3 – 3D time-lapse and tracking – live hemocytes, drosophila pupae – Spinning Disk 2 (4h – 4 students)

• 4a – FRAP – GFP-tagged SFs, live HeLa cells - Point Scanning Confocal 1 (2h – 2 students)
• 4b – AP-FRET – YFP and mCherry-tagged SFs, fixed cells - Point Confocal 2(2h – 2 students)

The second module will consist of one morning of introductory lectures on the basics of image processing followed by hands-on exercises applied to data collected in the first module, performed by individual students.

• José Rino, PhD – BioImaging Unit, IMM
• António Temudo – BioImaging Unit, IMM
• Ricardo Henriques – Gene Expression and Biophysics Unit, IMM
• Soren Prag, PhD – Tissue and Morphogenesis Unit, IMM
• Ghislain Cabal, PhD – Molecular Malaria Unit, IMM

4 inverted and 1 upright microscopes prepared for live cell imaging:

• Andor Revolution spinning disk confocal
• Zeiss LSM 710 point-scanning confocal
• Zeiss LSM 510 META point-scanning confocal
• Zeiss Axiovert 200M widefield motorized epifluorescence microscope
• Zeiss LSM 5 Live line-scanning confocal

Registrations are already closed. Thank you for your interest.

For further course information please contact José Rino at joserino@fm.ul.pt

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